A cooperative response to endocardial NOTCH signaling stimulation regulates transcriptional activity during cardiac valve development and disease

Abstract

BackgroundThe endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve (BAV) formation and/or calcific aortic valve disease (CAVD), but knowledge is very limited about the role played in valve development and disease by non-coding endocardial regulatory regions and upstream factors. MethodsWe manipulated the NOTCH signaling pathway in mouse embryonic endocardial cells by short-term and long-term co-culture with OP9 stromal cells expressing NOTCH ligands and treatment with the {gamma}-secretase inhibitor RO4929097, defining the transcriptional profile associated to each condition. The endocardial chromatin accessibility landscape for each condition was defined by high-throughput sequencing (ATAC-seq) determination of transposase-accessible chromatin. In vitro and in vivo models carrying deletions of different non-coding regulatory elements were generated by CRISPR-Cas9 gene editing. ResultsWe identified primary and secondary transcriptional responses to NOTCH ligands in the mouse embryonic endocardium. By integrating our gene expression data with data from developing valves of mice with NOTCH loss-of-function and from human valve calcification samples, we were able to identify a NOTCH-dependent transcriptional signature in valve development and disease. Further, by defining the endocardial chromatin accessibility landscape after NOTCH pathway manipulation and integrating with in vivo data from developing mouse endocardium and adult human valves, we were able to identify a set of potential non-coding regulatory elements, validate representative candidates, propose co-factors interacting with them, and define the timeframe of their regulatory activity. Analysis of the transcriptional repression driven by NOTCH activation revealed cooperation between the NOTCH and HIPPO pathways in the endocardium during cardiac valve development. ConclusionsTranscriptional regulation in the embryonic endocardium after NOTCH pathway stimulation occurs in a sequential manner and requires the participation of several factors. NOTCH not only triggers the transcriptional activity of the non-coding elements recognized by these factors, but also represses those elements whose activity negatively affects the development and homeostasis of the cardiac valves. Novelty and SignificanceWHAT IS KNOWN? O_LIThe embryonic endocardium is a source of cell types and a crucial signaling center for cardiac valve development and maturation. C_LIO_LIEndocardial NOTCH pathway activity patterns specific cellular behaviors that will give rise to the cardiac valve primordia, guide their maturation, and maintain adult valve homeostasis. C_LIO_LINOTCH signaling abrogation is associated with human valve dysmorphology (bicuspid aortic valve) and adult calcific aortic valve disease (CAVD). C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE? O_LIThe existence of a primary transcriptional response after short-term NOTCH activation that is enhanced after sustained ligand stimulation, and a secondary response triggered after long-term NOTCH activation. C_LIO_LIWe have identified a NOTCH-dependent transcriptional signature specific for valve development and disease. C_LIO_LIWe have defined the endocardial chromatin accessibility landscape after NOTCH pathway manipulation, and proposed a set of potential cofactors and the time frame of their activity involved in the NOTCH-dependent transcriptional response. C_LIO_LIWe have identified a set of potential non-coding regulatory elements involved in valve development and disease, and evaluated them in vitro and in vivo. C_LIO_LIWe have identified the cooperation between the NOTCH and HIPPO pathways in the endocardium during cardiac valve development. C_LI